Abstract

Early detection and targeted therapy are two major challenges in the battle against cancer. Novel imaging contrast agents and targeting approaches are greatly needed to improve the sensitivity and specificity of cancer theranostic agents. Here, we implemented a novel approach using a magnetic micromesh and biocompatible fluorescent magnetic nanoparticles (FMN) to magnetically enhance cancer targeting in living subjects. This approach enables magnetic targeting of systemically administered individual FMN, containing a single 8 nm superparamagnetic iron oxide core. Using a human glioblastoma mouse model, we show that nanoparticles can be magnetically retained in both the tumor neovasculature and surrounding tumor tissues. Magnetic accumulation of nanoparticles within the neovasculature was observable by fluorescence intravital microscopy in real time. Finally, we demonstrate that such magnetically enhanced cancer targeting augments the biological functions of molecules linked to the nanoparticle surface.

Abstract

Particle manipulation based on dielectrophoresis (DEP) can be a versatile and useful tool in lab-on-chip systems for a wide range of cell patterning and tissue engineering applications. Even though there are extensive reports on the use of DEP for cell patterning applications, the development of approaches that make DEP even more affordable and common place is still desirable. In this study, we present the use of interdigitated electrodes on a printed circuit board (PCB) that can be reused to manipulate and position HeLa cells and polystyrene particles over 100 microm thick glass cover slips using DEP. An open-well or a closed microfluidic channel, both made of PDMS, was placed on the glass coverslip, which was then placed directly over the PCB. An AC voltage was applied to the electrodes on the PCB to induce DEP on the particles through the thin glass coverslip. The HeLa cells patterned with DEP were subsequently grown to confirm the lack of any adverse affects from the electric fields. This alternative and reusable platform for DEP particle manipulation can provide a convenient and rapid method for prototyping a DEP-based lab-on-chip system, cost-sensitive lab-on-chip applications, and a wide range of tissue engineering applications.

Abstract

We present a novel, on-chip system for the electrokinetic capture of bacterial cells and their identification using the polymerase chain reaction (PCR). The system comprises a glass-silicon platform with a set of micro-channels, -chambers, and -electrodes. A platinum thin film resistor, placed in the proximity of the chambers, is used for temperature monitoring. The whole chip assembly is mounted on a Printed Circuit Board (PCB) and wire-bonded to it. The PCB has an embedded heater that is utilized for PCR thermal cycle and is controlled by a Lab-View program. Similar to our previous work, one set of electrodes on the chip inside the bigger chamber (0.6 microl volume) is used for diverting bacterial cells from a flowing stream into to a smaller chamber (0.4 nl volume). A second set of interdigitated electrodes (in smaller chamber) is used to actively trap and concentrate the bacterial cells using dielectrophoresis (DEP). In the presence of the DEP force, with the cells still entrapped in the micro-chamber, PCR mix is injected into the chamber. Subsequently, PCR amplification with SYBR Green detection is used for genetic identification of Listeria monocytogenes V7 cells. The increase in fluorescence is recorded with a photomultiplier tube module mounted over an epifluorescence microscope. This integrated micro-system is capable of genetic amplification and identification of as few as 60 cells of L. monocytogenes V7 in less than 90 min, in 600 nl volume collected from a sample of 10(4) cfu ml(-1). Specificity trials using various concentrations of L. monocytogenes V7, Listeria innocua F4248, and Escherichia coli O157:H7 were carried out successfully using two different primer sets specific for a regulatory gene of L. monocytogenes, prfA and 16S rRNA primer specific for the Listeria spp., and no cross-reactivity was observed.

Abstract

Destruction of hypoxic regions within tumors, virtually inaccessible to cancer therapies, may well prevent malignant progression. The tumor's recruitment of monocytes into these regions may be exploited for nanoparticle-based delivery. Monocytes containing therapeutic nanoparticles could serve as "Trojan Horses" for nanoparticle transport into these tumor regions. Here we report the demonstration of several key steps toward this therapeutic strategy: phagocytosis of Au nanoshells, and photoinduced cell death of monocytes/macrophages as isolates and within tumor spheroids.

Abstract

Nanoparticles and bacteria can be used, independently, to deliver genes and proteins into mammalian cells for monitoring or altering gene expression and protein production. Here, we show the simultaneous use of nanoparticles and bacteria to deliver DNA-based model drug molecules in vivo and in vitro. In our approach, cargo (in this case, a fluorescent or a bioluminescent gene) is loaded onto the nanoparticles, which are carried on the bacteria surface. When incubated with cells, the cargo-carrying bacteria ('microbots') were internalized by the cells, and the genes released from the nanoparticles were expressed in the cells. Mice injected with microbots also successfully expressed the genes as seen by the luminescence in different organs. This new approach may be used to deliver different types of cargo into live animals and a variety of cells in culture without the need for complicated genetic manipulations.

Abstract

Solid-state nanopores have emerged as possible candidates for next-generation DNA sequencing devices. In such a device, the DNA sequence would be determined by measuring how the forces on the DNA molecules, and also the ion currents through the nanopore, change as the molecules pass through the nanopore. Unlike their biological counterparts, solid-state nanopores have the advantage that they can withstand a wide range of analyte solutions and environments. Here we report solid-state nanopore channels that are selective towards single-stranded DNA (ssDNA). Nanopores functionalized with a 'probe' of hair-pin loop DNA can, under an applied electrical field, selectively transport short lengths of 'target' ssDNA that are complementary to the probe. Even a single base mismatch between the probe and the target results in longer translocation pulses and a significantly reduced number of translocation events. Our single-molecule measurements allow us to measure separately the molecular flux and the pulse duration, providing a tool to gain fundamental insight into the channel-molecule interactions. The results can be explained in the conceptual framework of diffusive molecular transport with particle-channel interactions.

Abstract

The decrease in resonant frequency (-Deltaomega(r)) of a classical cantilever provides a sensitive measure of the mass of entities attached on its surface. This elementary phenomenon has been the basis of a new class of bio-nanomechanical devices as sensing components of integrated microsystems that can perform rapid, sensitive, and selective detection of biological and biochemical entities. Based on classical analysis, there is a widespread perception that smaller sensors are more sensitive (sensitivity approximately -0.5omega(r)/m(C), where m(C) is the mass of the cantilever), and this notion has motivated scaling of biosensors to nanoscale dimensions. In this work, we show that the response of a nanomechanical biosensor is far more complex than previously anticipated. Indeed, in contrast to classical microscale sensors, the resonant frequencies of the nanosensor may actually decrease or increase after attachment of protein molecules. We demonstrate theoretically and experimentally that the direction of the frequency change arises from a size-specific modification of diffusion and attachment kinetics of biomolecules on the cantilevers. This work may have broad impact on microscale and nanoscale biosensor design, especially when predicting the characteristics of bio-nanoelectromechanical sensors functionalized with biological capture molecules.

Abstract

Bacteriophage phi29 virus nanoparticles and its associated DNA packaging nanomotor can provide for novel possibilities towards the development of hybrid bio-nano structures. Towards the goal of interfacing the phi29 viruses and nanomotors with artificial micro and nanostructures, we fabricated nanoporous Anodic Aluminum Oxide (AAO) membranes with pore size of 70 nm and shrunk the pores to sub 40 nm diameter using atomic layer deposition (ALD) of Aluminum Oxide. We were able to capture and align particles in the anodized nanopores using two methods. Firstly, a functionalization and polishing process to chemically attach the particles in the inner surface of the pores was developed. Secondly, centrifugation of the particles was utilized to align them in the pores of the nanoporous membranes. In addition, when a mixture of empty capsids and packaged particles was centrifuged at specific speeds, it was found that the empty capsids deform and pass through 40 nm diameter pores whereas the particles packaged with DNA were mainly retained at the top surface of the nanoporous membranes. Fluorescence microscopy was used to verify the selective filtration of empty capsids through the nanoporous membranes.

Abstract

Surfaces of materials that promote cell adhesion, proliferation, and growth are critical for new generation of implantable biomedical devices. These films should be able to coat complex geometrical shapes very conformally, with smooth surfaces to produce hermetic bioinert protective coatings, or to provide surfaces for cell grafting through appropriate functionalization. Upon performing a survey of desirable properties such as chemical inertness, low friction coefficient, high wear resistance, and a high Young's modulus, diamond films emerge as very attractive candidates for coatings for biomedical devices. A promising novel material is ultrananocrystalline diamond (UNCD) in thin film form, since UNCD possesses the desirable properties of diamond and can be deposited as a very smooth, conformal coating using chemical vapor deposition. In this paper, we compared cell adhesion, proliferation, and growth on UNCD films, silicon, and platinum films substrates using different cell lines. Our results showed that UNCD films exhibited superior characteristics including cell number, total cell area, and cell spreading. The results could be attributed to the nanostructured nature or a combination of nanostructure/surface chemistry of UNCD, which provides a high surface energy, hence promoting adhesion between the receptors on the cell surface and the UNCD films.

Abstract

A probe array with nano-scale tips, integrated into a micro-fluidic channel was developed for the capture and lysing of small number of vaccinia virus particles using dielectrophoresis. The nano-scale probe array was fabricated in Silicon on Insulator (SOI) wafers, and sharpened with repeated oxidation steps. The gap between each probe ranged from 100 nm to 1.5 microm depending on fabrication parameters. The probe array was used to capture vaccinia virus using positive dielectrophoresis (DEP) from a flow within the microfluidic channel, and then the same probe array was used to apply high electric field to lyse the virus particles. It was shown that under electric field strengths of about 10(7)V/m, the permeability of ethidium bromide into the vaccinia virus particles was increased. Upon SEM analysis, the particles were found to be damaged and exhibited tubules networks, indicating disintegration of the virus outer layer. In addition, elongated strands of DNA were clearly observed on the chip surface after the application of the high electric field, demonstrating the possibility of electrical lysis of virus particles.

Abstract

This work describes a simple approach to immobilize functionalized colloidal microstructures onto a C(18)-coated SiO(2) substrate via specific or non-specific bio-mediated interactions. Biotinylated bovine serum albumin pre-adsorbed onto a C(18) surface was used to mediate the surface assembly of streptavidin-coated microbeads (2.8 microm), while a bare C(18) surface was used to immobilize anti-Listeria antibody-coated microbeads (2.8 microm) through hydrophobic interactions. For a C(18) surface pre-adsorbed with bovine serum albumin, hydrophobic polystyrene microbeads (0.8 microm) and positively charged dimethylamino microbeads (0.8 microm) were allowed to self-assemble onto the surface. A monolayer with high surface coverage was observed for both polystyrene and dimethylamino microbeads. The adsorption characteristics of Escherichia coli and Listeria monocytogenes on these microbead-based surfaces were studied using fluorescence microscopy. Both streptavidin microbeads pre-adsorbed with biotinylated anti-Listeria antibody and anti-Listeria antibody-coated microbeads showed specific capture of L. monocytogenes, while polystyrene and dimethylamino microbeads captured both E. coli and L. monocytogenes non-specifically. The preparation of microbead-based surfaces for the construction of microfluidic devices for separation, detection, or analysis of specific biological species is discussed.